Cardiovascular Pharmacology of CPU-23: A Novel Calcium Channel Blocker

Cardiovascular Drug Reviews Vol. 14, No. 4, pp. 36&379 0 1996 Neva Press, Branford. Connecticut

Cardiovascular Pharmacology of CPU-23: A Novel Calcium Channel Blocker Hui Dong, Gareth J . Waldron, William C. Cole, and Christopher R. Triggle Faculy of Medicine, University of Calgan, Alberta, EN 4NI Canada

Key Words: Antiarrhythmie-Antihypertensive-Calcium sion--CPU-23-Vasodilation.

channel blockerXardiac depres-

INTRODUCTION The use of calcium channel blockers has become increasingly popular for the treatment of hypertension, angina pectoris, supraventricular arrhythmia, and several other cardiovascular diseases. Currently, three distinct classes of calcium channel blockers of the L-type calcium channel are in clinical use, namely the dihydropyridines (e.g., nifedipine, nitrendipine and nimodipine), the phenylalkylamines (e.g. , verapamil), and the benzothiazepines (e.g., diltiazem) (5). The prototypic dihydropyridine calcium channel blocker, nifedipine (Fig. I ) , can induce vasorelaxation and lower blood pressure by reduction of peripheral vascular resistance (7,12). It also produces a reflex tachycardia, however, which somewhat limits its clinical use. While numerous new dihydropyridine analogs are currently being developed by different laboratories to provide better benefit-to-risk ratios than nifedipine (20), we and others have been developing novel calcium channel blockers based on lead compounds isolated from Chinese medicinal herbs (1 1,17). Tetrandnne is an alkaloid isolated from the Chinese medical herb Radix stephania tetrandrae. It has been used clinically in China to treat angina and hypertension with good results ( 15). Recent pharmacological studies have demonstrated that tetrandrine blocks inward calcium current through the voltage-sensitive L-type calcium channel (19). Since it competitively inhibits [ 3H]diltiazem binding, partly inhibits [3H]verapamil binding, and stimulates [3H]nitrendipine binding to cardiac sarcolemmal membranes in a manner very similar to diltiazem, it is likely that tetrandrine acts at the benzothiazepine site of the L-type calcium channel (18). Chemically, tetrandrine is a bis-benzyl-tetrahydroisoquinolinewith its two subunits connected in a head-to-head, tail-to-tail fashion through ether linkages (Fig. 1). Chemical synthesis of tetrandnne is difficult because of its complicated structure. The pharmacological profile of tetrandrine indicates that it has less potency and selectivity for blood vessels than do the dihydropyridine calcium channel blockers (12). The clinical use of Address correspondence and reprint requests to Dr. Hui Dong, Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada. Fax: 403-270-2211 .

364

CPU-23

365

3

‘C H3

0

0

OCH,

CPU-23.

y&CWMe

MeOOC Me

Me

H

Nifedipine FIG. 1. Chemical structures of tetrandrine, CPU-23. and nifedipine. Note: CPU-23 is the equivalent of a half-molecule of tetrandrine.

tetrandrine has, therefore, been somewhat limited. In order to simplify the chemical structure of tetrandrine and find better therapeutic agents, we have synthesized and screened, using both [3H]nitrendipine binding and biological assays, a series of benzyltetrahydroisoquinolines (half-molecules of tetrandrine). CPU-23, the most potent com-

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pound identified in this series, inhibited KC1-induced contraction of rat aorta and displaced [3H]nitrendipine binding in rat cerebral cortical membranes with similar potency. CPU-23 also behaved as a simple competitive inhibitor at the [3H]nitrendipine binding site (8). In addition, it inhibited KC1-induced Ca2+ influx and action potential characteristics of myocardial preparations in vitro and induced hypotension and bradycardia in rats in vivo. It was suggested, therefore, that CPU-23 may exert its cardiovascular effects by inhibiting calcium influx via specific calcium channels, probably L-type calcium channels (8,9). This review summarizes in detail only the preclinical pharmacology of CPU-23, mainly cardiovascular pharmacology.

CHEMISTRY CPU-23 was synthesized by The Division of Medical Chemistry, China Pharmaceutical University. The chemical name of CPU-23 is 1-{ 1-[(6-methoxy)-naphth-2-yl]}-ethyl-2(1-piperidinyl)-acetyl-6,7-dimethoxy- 1,2,3.4-tetrahydroisoquinoline(Fig. 1). The compound is a light yellowish, white crystalline powder with a melting point of 137-138"C, the molecular weight is 502.65. CPU-23 is highly soluble in ethanol and slightly soluble in water, but CPU-23 . HCl is highly soluble in water. CPU-23 is stable at room temperature.

IN VITRO PHARMACOLOGY [3H]Nitrendipine Binding Studies With the availability of radioactive ligands of dihydropyridines, verapamil, and diltiazem with high specific activity, it has become possible to study drug interactions at the L-type calcium channel by direct radioligand binding assays. Such studies indicate that these compounds interact at distinct binding sites that are allosterically linked to the channel complex and that occupation of one site affects ligand binding at other sites. For instance, the specific binding of [3H]nitrendipine to cardiac sarcolemma and brain membrane was competitively inhibited by nifedipine, only partly inhibited by verapamil, and allosterically stimulated by diltiazem or tetrandrine (6,18). Unexpectedly, in our 13H]nitrendipine binding study, none of the substituted tetrahydroisoquinolines exhibited a stimulatory effect, unlike tetrandrine, which stimulated [3H]nitrendipine binding. Instead, many of them inhibited [3H]nitrendipine binding to rat cerebral cortical membranes in a concentration-dependent manner (Table 1). From the relative potencies of substituted tetrahydroisoquinolines in inhibiting [3H]nitrendipine binding, several structure-activity features can be deduced (for details see ref. 8). CPU-23 was one of the most potent of the tetrahydroisoquinoline analogues identified in this series, with an IC,, value of 0.51 FM.When the displacement curve was analyzed with the Hill plot, the Hill coefficient was found to be close to unity, suggesting that CPU-23 may act as a simple competitive inhibitor at the nitrendipine binding site of the L-type calcium channel. To evaluate further the nature of the interaction with the dihydropyridine site, we analyzed the effects of various concentrations of CPU-23 on the K, (equilibrium dissociation constant) and B,, (maximum number of binding sites) of r3H]nitrendipine binding by saturation analysis. As illustrated in Table 2, CPU-23 be-

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TABLE 1. Inhibition of specific [3H]nitrendipine binding to rat cerebral cortical membranes and 80 mM KCI-induced contraction of rat aortic strips by classical calcium entry blockers and tetrahydroisoquinolines

[3H]nitrendipine binding

Drugs Nifedipine Verapamil Diltiazem Tetrandrine CPU-21 CPU-23 CPU-50 CPU-57

Contraction of rat aortic strips

1.1 t 0.2 x 10-9 2.1 t 0.1 x 1 0 - ~ Stimulation* Stimulation* 4.6 2 0.6 X 5.1 2 0.8 X 7.6 t 1.0 x 10-7 4.1 t 0.1 x 1 0 - ~

1.2 t 0.3 x 6.3 2 0.5 X lo-' 1.4 0.2 x 10-7 7.0 2 1.9 X 2.5 2 0.3 X 2.1 2 0.2 x 5.0 t 0.4 X 2.5 t 0.3 X

IC,, is the molar concentration of drug required to give 50% inhibition of specific [3H]nitrendipinebinding or 80 mM KCI-induced contraction of rat aortic strips. Results are the mean 2 S, of 3 (for binding) and 6 (for M) and tetrandrine (lo-' M) enhanced specific [3H]nitrenbioassay) separate experiments. *Diltiazem dipine binding at 37°C to 168 t 8% and 263 ? 29% of control, respectively. The same concentration of diltiazem did not stimulate I3H]nitrendipine binding at 22°C (103 t 3%) while tetrandrine was still able to enhance binding to 154 -+ 6% of control. Reprinted from ref. 8 with permission.

haved as a simple competitive inhibitor like nifedipine and significantly reduced the apparent affinity without affecting the B,, of [3H]nitrendipinebinding. When the original data for CPU-23 were further analyzed by the Arunlakshana & Schild plot, a slope close to unity (0.94) and a K, value of 0.17 pM were obtained. This K, value agrees quite well with the Ki value (0.26 pM) calculated from the drug displacementexperiments by the Cheng-Prusoff equations (8). Further experiments to examine the effects of CPU23 on the dissociation kinetics of [3H]nitrendipine will provide a clear answer to the competitive nature of this interaction.

Effects on Vascular Smooth Muscle In a similar manner to that of the other prototypic calcium channel blockers, CPU-23 produced concentration-dependent inhibition of the contraction of rat aorta induced by 80 TABLE 2. Scatchard analysis of the interaction of CPU-23 with the binding of [3H]nitrendipine to rat cerebral cortical membranes Conc

(M) 0 3 x 10-7 lor6

0.31 t 0.03 (6) 0.48 t 0.07 (4)* 1.15 t 0.09 (3)** 1.81 t 0.20 (3)**

B, (fmol mg-' protein)

Hill coefficient (nd

18.23 t 1.15 (6) 16.49 2 1.45 (4) 20.11 2 2.74 (3) 15.47 t 1.79 (3)

0.81 t 0.04 (6) 0.83 0.04 (4) 0.75 2 0.09 (3) 1.00 ? 0.05 (3)

*

['Hlnitrendipine (0.06-3 nM) was incubated with rat cerebral cortical membranes in the presence or absence of CPU-23 for 30 min at room temperature and specific binding was measured. K, and B,,, were determined by Scatchard analysis with the LIGAND computer programme (Elsevier-BIOSOFT). Data shown are the mean 2 SF of (n)separate experiments performed in duplicate. *P < 0.05, **P < 0.001 when compared with control. Reprinted from ref. 8 with permission.

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H . DONG ET M .

mM KCl with a pD, value of 6.68. The rank order of potencies in this bioassay was: nifedipine > verapamil > diltiazem 3 CPU-23 > tetrandrine. Although CPU-23 was 175 times less potent than nifedipine, it was about 3 times more potent than tetrandrine in this test (Table 1). There was a good correlation (r = 0.99, p < 0.001) between the potency of substituted tetrahydroisoquinolines to inhibit specific [3H]nitrendipine binding to rat cerebral cortical membranes and their ability to inhibit KC1-induced contraction of rat aorta (Fig. 2). This further supports the notion that substituted tetrahydroisoquinolines relax arterial smooth muscle by blocking L-type calcium channels through interaction with the dihydropyridine site. In isolated guinea pig middle cerebral artery (in the presence of NG-nitro-L-arginine and indomethacin to prevent the production of NO and cyclo-oxygenase metabolites in endothelial cells), CPU-23 produced concentration-dependentrelaxation of 80 mM KC1or histamine- induced contraction, with pD, values of 6.60 and 7.04, respectively. When compared with CPU-23, nimodipine, a dihydropyridine with high selectivity for cerebral arteries, was more potent in the same tissue, with pD, values of 9.06 and 9.48 for relaxing 80 mM KCl- and histamine-induced contractions, respectively (H. Dong, unpublished data). The spontaneous contractions of the rat portal vein were dependent on extracellular calcium as they were abolished in calcium-free or 2 mM EGTA-containing physiological solutions and were restored upon readministration of 2.5 rnh4 Ca2' or washout of EGTA. CPU-23 shared with verapamil and diltiazem the ability to inhibit the spontaneous contractions of the rat portal vein in a concentration-dependent manner (H. Dong, unpublished data). These data suggest that the vasorelaxant activity of CPU-23 is not limited

r = 0.993( P < 0.001)

Nifedipine

/

-log

Binding

FIG. 2. Correlation between antagonism of 80 mM KCI-induced contraction of rat isolated aortic strips and inhibition of specific [ 3H]nitrendipinebinding by nifedipine and tetrahydroisoquinolines.The correlation coefficient (r = 0.99, P < 0.001) was calculated by linear regression analysis. Reprinted from ref. 8 with permission.

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to large peripheral arteries and not dependent upon endothelial-derived mediators, but is effective in cerebral resistance vessels and results from the inhibition of extracellular calcium entry into the vascular smooth muscle cells.

Cardiac Effects In Langendorff perfused rat hearts, CPU-23, nifedipine, or diltiazem decreased the heart rate in a dose-dependent manner; nifedipine was less potent than the other two drugs. Both nifedipine and diltiazem markedly inhibited the force of contraction in a concentration-dependent manner. On the other hand, the effect of CPU-23 was biphasic, inhibiting the developed tension at concentrations of 0.1-1 pM but enhancing the tension at 10 pM (Fig. 3). Nifedipine possessed a more potent negative inotropic effect than negative chronotropic effect and caused cardiac arrest at concentrations higher than 0.3 pM. Interestingly, CPU-23 had a more potent negative chronotropic effect than negative inotropic effect and increased cardiac contraction at concentrations higher than 10 pM. In electrophysiological studies, CPU-23 decreased the action potential amplitude (APA) and maximum upstroke velocity (Vmax)and shortened the action potential duration at 50% and 90% of repolarization (APD,, and APD,) of action potentials in guinea pig papillary muscles. These effects were observed only at a concentration of 100 pM. CPU-23 had no effect on resting potential (RP) or overshoot (0s)(Table 3). CPU-23, in and APD,, of slow a dose-dependent manner (1-100 pM),decreased APA, 0s and V, action potentials induced by histamine in K+-depolarized guinea pig papillary muscle, where the action potential configuration was very dependent on calcium entry (Table 4

Control

3 110-8

10-8

I 0-7

3

3

I

10-6

10-5

3

I

10‘‘

10-5

I

10-7

.

Nifcdipinc [ h l )

Control

10-7

3

10-7

10-6

Diltiarernlhl)

Control

I 0-7

3

I

lo-’

10-6

CI’UZ3 lhll

FIG. 3. Typical tracings of the actions of nifedipine, diltiazem, and CPU-23 on heart rate and myocardial developed tension in Langendorff perfused heart of rats. The spontaneously beating hearts were allowed to adapt to the new environmental conditions for 20 min before the administration of drugs from the low to high concentrations. Chart speeds: 50 mdmin (slow) or 10 m d s e c (fast).

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370 TABLE 3. Effects of

M CPU-23 on fast action potential characteristics in guinea pig

papillay muscles

89.3 88.8

Control CPU-23

2 ?

0.9 1.0

0s

v,,,

APA (mV)

(mV)

APDm (ms)

APD, (ms)

(VS - ')

135.5 2 2.2 126.0 2 1.4*

46.3 2 2.0 37.3 2 1.9*

151.1 2 13.6 143.8 2 12.6*

200.8 2 10.4 191.3 2 10.2*

202.5 -t 1.8 183.5 2 8.6*

RP (mV)

Data shown are the mean 2 S, of 4 separate experiments. RP:resting potential; APA: amplitude of action potential; 0s: overshoot; APD,, and APD,: action potential duration at 50 and 90% of repolarization, respectively; V,,,: maximum upstroke velocity: *P < 0.05 vs control. Reprinted from ref. 9.

and Fig. 4).In rabbit sinoatrial node tissue, where the action potential configuration was also dependent on calcium entry, CPU-23 decreased the APA, spontaneous rate, and slope of phase 4 depolarization (dVldt) in a dose-dependent manner (Table 5). KCl increases cytosolic free Ca2 concentration [Ca2'1, in fura-2 loaded rat ventricular myocytes in a dose-dependent manner, only in a medium containing calcium; this effect is attenuated by nifedipine. In contrast, caffeine increased [Ca2+],even in the absence of extracellular calcium. This suggests that the KC1-induced increase in [Ca2+Iiis due to extracellular calcium influx through L-type calcium channels, whereas the caffeineinduced increase in [Ca2+],is dependent on intracellular Ca2+ mobilization. CPU-23 (1-10 kM) significantly inhibited the KC1-induced increase in [Ca2+],of rat ventricular myocytes (Fig. 6), but had no significant effect on the caffeine-induced increase in [Ca2+],of rat ventricular myocytes in the calcium-free medium (Fig. 5). These findings provide direct evidence that CPU-23 selectively inhibits calcium entry in ventricular myocytes. +

Effects on Tracheal Smooth Muscle In guinea pig isolated tracheal smooth muscle, CPU-23 relaxed the tension produced by high K + , histamine, or PGF,, in a dose-dependent manner, with pD, values of 7.39, 7.21, and 7.28, respectively. When isolated tracheal smooth muscle is depolarized by high K , subsequent administration of Ca2+ induces contraction in a dose-dependent +

TABLE 4. Effects of CPU-23 on slow action potential characteristics in guinea pig Daoillan muscles

Control CPU-23 M lo-' M lo-" M

56.2

2

5.4

94.6

54.8 53.0 50.7

-t

5.7 3.0 3.1

84.4 t 2.9* 73.0 2 2.6* 67.0 2 6.5**

2 2

2

4.1

177.0 2 13.2

11.O

28.8

168.6 2 11.8* 145.0 2 3.0* 84.3 2 4.3**

10.2 2 0.6* 7.3 -t 0.3** 4.3 ? 0.6**

5

4.3*

21.7 2 3.9%

17.7 2 3.9**

Data shown are the mean 2 S, of 5 separate experiments. MDP: maximum diastolic potential; APA: amplitude of action potential; 0s: overshoot; APD,: potential duration at 90% of repolarization; Vmx: maximum upstroke velocity. * P < 0.05; **P < 0.01 vs control. Reprinted from ref. 9 with permission.

Cordiovasculor Drug Revieus. Vol 14. No. 4 .

1996

0.5

33.0 2 3.1

2

action

CPU-23

371

0

>

-

E

-60

1' I

looms

FIG. 4. Typical tracings of the effects of CPU-23 on slow action potential of guinea pig papillary muscles induced by 22 mM K + and M histamine. A: control; B: CPU-23 M; C: CPU-23 M; D: CPU-23 M. Driving rate: 0.2 Hz.

manner due to extracellular calcium influx through voltage-operated calcium channels (VOC). CPU-23 (0.014.1 pM) significantly depressed this dose-response curve for Ca2+ with a pDi value of 7.37. CPU-23 also depressed the dose-response curve for acetylcholine and histamine on tracheal smooth muscle with pD, values of 6.92 and 5.88, respectively (H. Dong, unpublished data). Compared with the other calcium channel blockers in the same preparation, the inhibitory potency of CPU-23 is close to the dihydropyridine derivatives, such as nifedipine and greater than that of verapamil or diltiazem (1). In our experiments in vivo, we also observed that CPU-23 significantly increased the respiratory intensity and frequency of rats after intravenous (i.v.) injection (H. Dong, unpublished data). Taken together, these TABLE 5. Effects of CPU-23 on maximum diastolic potential (MDP), amplitude of action potential ( M A ) , spontaneous rate (SR) and slope of phase 4 spontaneous depolarization (dVldt) of rabbit sinoatrial node tissue MDP (mV) Control CPU-23 lo-' M M 3 X 10-6M

51.8

f

3.1

52.4 f 2.9 48.0 f 1.8 41.8 -+ 4.0

PA (mV)

SR (b.p.m.)

38.5 2 3.4

206

37.5 f 3.1 32.3 f 4.4* 25.0 f 1.1*

180? 5* 166 ? lo** 97 f 18**

f

10

dVidt (mv S C ' )

53.0 f 12.0 42.0 38.0 26.8

f 11.8* f 10.7*

*

6.8*

Data shown are the mean i S , of 5 separate experiments. *P < 0.05; **P < 0.01 vs control. Reprinted from ref. 9 with permission.

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3 72

H

c

-

"

n

0

0

**

100

Y

0

FIG. 5. Effects of caffeine on cytosolic calcium concentration ([Ca' *Ii)and CPU-23 on caffeine-induced [Ca'*], increase in rat ventricular myocytes in Ca"-free solution. (0)Control; (m) Caffeine 10 mM; (W) CPU23 M pretreatment for 30 min before caffeine 10 mM added. Each bar is the mean of 6 separate experiments with S,. **P < 0.01 when compared with control.

findings suggest that CPU-23 may have potential therapeutic significance in some respiratory disorders, such as asthma and pulmonary hypertension.

IN VIVO PHARMACOLOGY Hypotensive Effects after Intravenous Injection In pentobarbital-anesthetized Sprague-Dawley (SD) rats, acute administration of CPU-23 (1-10 mg/kg i.v.) caused a rapid-onset and dose-dependent decrease in mean arterial blood pressure (MAP) and heart rate (HR). The hypotensive and bradycardic

100

-

b

80

-

--

60-

10

0

40-

4

20-

2 E

c

n

.-C

it.

00

-

r', .

,

. , . , .

20

4C

60 80 KCI (mM)

100

120

140

FIG. 6. Inhibitory effects of CPU-23 on KCI-induced increase in [Ca"], in rat ventricular myocytes in 1.25 mM Ca" solution. Each point is the mean of 5 separate experiments with S , . *P < 0.05; **P < 0.01, when compared with control. Baseline [Ca' 1, level: 128 2 1 1 nM. (0) Control; CPU-23 M; (8)CPU-23 M. Reprinted from ref. 9.

(e)

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CPU-23

effects were rather slight and short-lived at 1 mg/kg, but more pronounced and longer lasting at 3-10 mg/kg at the higher concentrations, MAP and HR remained significantly depressed even at 60 min after drug administration (Fig. 7). Nifedipine, at a dose of 0.3 mg/kg i.v., produced a decrease in MAP comparable to that produced by 5 mg/kg CPU-23, indicating that CPU-23 is about 20 times less potent than nifedipine at lowering blood pressure in SD rats. CPU-23 is, however, about twice as effective as tetrandrine at lowering blood pressure (21). Interestingly, when compared to nifedipine, CPU-23 was over 500 times less potent at inhibiting [3H]nitrendipine binding and was 175 times less potent in relaxing high K+-induced tension of rat aorta in vitro, but was only 20 times less active in lowering rat blood pressure in vivo. These observations suggest that CPU-23 may have better bioavailability than nifedipine in vivo or may induce hypotension via other modes of action. CPU-23 also differed from nifedipine in its effect on HR. At doses that caused a similar degree of hypotension, CPU-23 exerted a pronounced negative chronotropic action, whereas nifedipine induced a reflex tachycardia. The marked bradycardia produced by CPU-23 may offer some myocardial protection in treating hypertensive conditions accompanied by a high circulatory concentration of catecholamines (22). Similar to the observations in SD rats, intravenous bolus injection of CPU-23 produced a

* I

I

I

1

I

0 1 3 5 10

I

20

30

b

40

1

1

50

60

6-6-8

Y

1

1

1

1

0135

I

10

I

I

20 30 Time ( m i d

I

I

I

40

50

60

FIG. 7. Effects of CPU-23 i.v. on (a) mean arterial blood pressure and (b) heart rate in pentobarbitalanaesthetized Sprague-Dawleyrats. The drug (1 mgkg (O), 3 mg/kg (0)and 10 mg/kg (0)) was administered at 0 min. Each point represents the mean of 5 animals; vertical bars show S,. *P < 0.05 when compared with control at zero time. Baseline: MAP = 105 2 5 mmHg, HR = 356 2 12 beatsimin. Reprinted from ref. 8 with permission.

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3 74

-10

0

10

20

30

40

60

50

TIME (Min) FIG. 8. Effects of CPU-23 (0.5 mgikg i.c.v.) on mean arterial blood pressure (MAP) in pentobarbital-

(m)

anesthetized Sprague-Dawley rats with or without bilateral cervical vagotomy. (0) Saline; CPU-23 with bilateral cervical vagotomy; (0) CPU-23 without bilateral cervical vagotomy. Vagotomy was performed 1 hr before drug administration. Injection of CPU-23 at time 0. Each point represents the mean of 6 animals; vertical b ~ show s S,. *P < 0.05: Significantly different when compared with the CPU-23-treated group. Reprinted from ref. 13 with permission.

a dose-dependent hypotensive and bradycardic response in both pentobarbitalanaesthetized spontaneously hypertensive (SHR) and normotensive WKY rats. The extent and duration of the hypotension or bradycardia induced by CPU-23 were similar in SHR and WKY rats.

TABLE 6. ECG changes after CPU-23 (I0mglkg i . v . ) in spontaneously hypertensive (SHRJ and normotensive WKY rats

P wave (mV) J point (mV) PP interval (ms) PR interval (ms)

SHR

WKY

0.16 2 0.06 0.40 t 0.07 0.38 0.09 0.06 t 0.02

0.09 ? 0.02* 0.15 2 0.06** 0.19 t 0.06** 0.02 2 0.01*

*

Data shown are the mean 2 S, of 5 separate experiments. *P < 0.05; **P < 0.01 vs SHR rats. Reprinted from ref. 10 with permission.

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TABLE 7 . EfSects of intravenous CPU-23 on early post-infection arrhythmias and mortality in Dentobarbital-anaesthetized rats VT

Group

n

Total numbers of ectopic beats

Saline CPU-23 1 mgkg-’ 2.5mgkg-’ 5mgkg-’

10

915 2 197

93 t 22

264 t 130** 40 2 18** 3 t 1**

37 2 26 2 2 2** 2 2 2**

8 6 8

VF

Duration

Incidence

Duration

Incidence

Mortality

(S)

(%I

(S)

(%)

(% in 30 min)

53 2 27

100

63 33** 13**

2

5

2**

0** 0**

70

20

13* O* * 0**

13 0** 0**

VT: ventricular tachycardia; VF: ventricular fibrillation. Data shown are the mean 2 S, of (n) animals. *P < 0.05; **P < 0.01 when compared with control. Reprinted from ref. 8 with permission.

Hypotensive Effects after Intracerebroventricular Injection Although the cardiovascular effects of calcium channel blockers are attributed mainly to a direct peripheral vasodilation and cardiac depression, recent findings suggest that they can exert indirect cardiovascular effects via interaction with the dihydropyridine receptor sites in the brain (3,4). CPU-23, at doses of 0.2-0.5 mglkg, which caused no significant cardiovascular effects after i. v. injection, induced a slow-onset, long-lasting and dosedependent decrease of MAP and HR after intracerebroventricular (i.c.v,) injection. This effect was similar to the response to nifedipine at a dose of 0.05 mgkg (13). The hypotensive effect of CPU-23 (0.5 mg/kg) was significantly attenuated (Fig. 8) in animals with bilateral cervical vagotomy, suggesting that these effects of CPU-23 are most likely of central origin, as were those of DHP derivatives in the same preparation (4,16). A large number of DHP receptor sites have been found in the brain and they have physiological roles in the regulation of cardiovascular activities. Calcium channel blockers may produce an excitatory effect of the nucleus tractus solitarius when administered centrally, resulting in hypotension and bradycardia in rats (16). Considering that the hydrophobic parameter (the ratio value of hydrophobic surface area of the molecule to the hydrophilic) of CPU-23 is close to that of nifedipine (0.546 vs. 0.508) (24), it is reasonable to infer that CPU-23 may pass through the blood-brain barrier to affect central regulation of cardiovascular activities as do the DHP derivatives. Interestingly, tetrandrine at the effective i.v. dose did not cause significant cardiovascular effects after i.c.v. injection (21), suggesting that CPU-23 may have advantages over tetrandrine in the treatment of hypertension.

Effects on Electrocardiogram CPU-23 (3-10 mglkg i.v.) slowed Ca2+-dependent sinus discharge rate and AV conduction as reflected by the frequency of P wave and the duration of PR interval in electrocardiograms (ECG) (14) in a dose-dependent manner, but did not alter Na+dependent intraventricular impulse propagation as reflected by the duration of QRS complex in SHR and WKY rats. These results suggest that CPU-23 exerts a calcium antagonism on the cardiac electrophysiological activity of rats in vivo (10). A comparison of CPU-23-induced ECG changes in different rats found that the effects on cardiac electric

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-

OJ

3OOJ control

FIG. 9. Simultaneous recordings of left ventricular pressure (LVP), their first derivatives over time (dPldt), arterial blood pressure (BP) and heart rate (HR) when the increasing dose of CPU-23 was intravenously injected into pentobarbitone-anaesthetized Sprague-Dawley rats. Chart speeds: 10 rndrnin (slow) or 10 m d s e c (fast).

activity were stronger in SHR than in WKY rats (Table 6). Since calcium channel blockers have been reported to be more potent as relaxants of vascular smooth muscle in SHR than in WKY rats (2), our finding suggests that calcium channel blockers may also exert a greater inhibition of the cardiac electric activity in hypertensive rats than in normotensive rats. The above-described characteristics of CPU-23 may be beneficial in the treatment of hypertension with some cardiac disorders.

Antiarrhythmic and Antiischaemic Effects Calcium channel blockers have been used in the treatments of arrhythmia, especially supraventricular arrhythmia, with good clinical results (23). As noted above, CPU-23 inhibits cytosolic Ca2+ increase in ventricular myocytes, the pacemaker action potentials in sinoatrial node, and the slow action potential of the papillary muscle in vitro. CPU-23 produces bradycardia and has more pronounced effects on ECG of hypertensive rats than those of normotensive rats in vivo. These cardiac effects of CPU-23 in vitro and in vivo suggest that it may have an antiarrhythmic action. Actually, CPU-23 reduced the total number of ectopic beats and both the duration and the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) occurring in the first 30 min following coronary artery ligation of rats in a dose-dependent manner. CPU-23 also reduced mortality due to ischemia reperfusion in a dose-dependent manner. At 2.5-5 mglkg i.v., CPU-23 abolished completely VF, which was accompanied by 100% survival (Table 7). These findings indicate that CPU-23 exerts antiarrhythmic and antiischaerllic effects like other calcium channel blockers.

Cardiovusculur Drug Reviews, Vol. 14, No. 4 . 1996

104 2 9

97 71 53

104 f 9

106 f 7 97 f 4 99 2 5

n

10

8 6 8

Saline CPU-23 1 mg/kg 2.5 mgikg 5 mg/kg

f f f

5 9** 4**

- 10

f

10

0

f f f

f

6** 32* 22**

10

103 f 7 79 f 5* 76 f 6*

104 f 9

409 323 283

454

0

76 70 66

71

*

f

f

f

1

423 ? 8* 348 f 20** 318 f 25**

455 2 9

3

41'1 5 5

7tt 72 f 4 t t 73 f 4 71 2 6

87 2 9 t t

3

MAP (mmHg) Time (min)

423 f 8* 323 f 32* 311 f 23**

454f 10

1

450

81 f 9 t 79 f 3 78 f 7

92 2 9 t t

5

f

9

459 f 6

20

459 f 6

30

9tt

105 f 9 86 f 9 86 2 9

93 2 8 t t

20

f

8t? 109 f 8 88 f 8 88 2 8

94

30

7 435 f 7 435 f 8 17** 365 f 18** 386 f 13** 13** 363 2 17** 373 f 15**

10

f f 5

2

10

89 t I0 82 f 2 83 5 9

92

423 f 8* 433 357 f 17** 357 3 1 9 2 25** 351

456 2 9

5

< 0.01 vs 0 min. Reproduced from reference 9 with permission.

101 t 5 78 f 5* 69 f 5**

105 f 9

-5

403 f 3** 313 f 28** 276 2 22**

452

-5

Values are mean f S, of (n) animals. * P < 0.05; **P < 0.01 vs control and t P < 0.05; t t P

-15

10

lo** 31** f 23**

f f

f

Treatment

471 f 11 403 453 f 12 288 430 2 12 243

10 450

- 10

8 6 8

f

- 15

10 454

n

Saline CPU-23 1 mgikg 2.5 mgikg 5 mgkg

Treatment

HR (beatdmin) Time (min)

subjected to coronary artery occlusion (at 0 min)

TABLE 8. Effects of CPU-23 (given 15 min before ligation) on heart rate (HR) and mean arterial blood pressure (MAP) in anaesthetized rats

LJ

E

B

3 78

H . DONG ETAL..

Hemodynamic Studies

When administered to anesthetized SD rats, CPU-23 (2.5-5 mgkg i.v.) produced a dose-dependent decrease in left ventricular pressure (LVP), the first derivative over time (dP/dL,), pulsatile blood pressure (BP), and heart rate (HR) (Fig. 9). The above hemodynamic effects appeared quickly and the peak effects were reached within 30 s after drug administration. At 5 mgkg, CPU-23 decreased the systolic blood pressure (SBP) by 39%, by diastolic blood pressure (DBP) by 4 1%, LVP by 30%, dP/dt,,, by 38%, - dP/dt,, 46%, and HR by 20%. Most of the hemodynamic parameters recovered in 30 mn, but HR and DBP remained significantly depressed even at 60 min after drug administration. To systematically compare the hernodynamic effects of CPU-23 with classical calcium channel blockers, nifedipine, verapamil, and diltiazem were selected as standards and administered at doses that produced a similar hypotensive effect, namely nifedipine 0.3 mg/kg, verapamil 1 mgkg, diltiazem 2 mgkg, and CPU-23 5 mgkg. At those doses, the rank order of effect in decreasing LVP and dPIdt,,,,, was verapamil > CPU-23 = diltiazem > nifedipine; the order in lowering HR was CPU-23 = verapamil = diltiazem > nifedipine (H. Dong, unpublished data). In ischemic arrhythmic rats, we observed that, although coronary artery ligation significantly lowered the arterial blood pressure, it did not further reduce the MAP in rats pretreated with CPU-23 at doses of 2.5-5 mg/kg (Table 8). Because CPU-23 did not further reduce MAP after coronary artery ligation at doses that produced significant antiarrhythmic action and reduced MAP of normal rats, it is possible that CPU-23 may be beneficial to patients with cardiac arrhythmias, as arterial blood pressure would not drop further with the use of the drug during myocardial ischemia.

+

SUMMARY The results of our studies conducted at the molecular, cellular, tissue, organ, and whole animals levels suggest that CPU-23 is a novel calcium channel blocker. This is further confirmed by recent studies utilizing the path-clamp technique, in which CPU-23 (1-10 pM) dose-dependently blocked inward Ca2+ currents via L-type CaZ+channels in single smooth muscle cells isolated from the rat tail artery (H. Dong & M. L. Earle, unpublished data). Although derived from tetrandrine, CPU-23 interacts at distinct binding sites on L-type calcium channels and exerts a more potent biological action than tetrandrine. These data suggest that CPU-23, because of its novel and unique chemical structure, may be a useful tool for the study of L-type calcium channels. The obvious antihypertensive and antiarrhythmic actions of CPU-23 and its respiratory effects indicate that CPU-23 may be a useful therapeutic agent worthy of further study and development. One weakness of CPU-23 is that its potency and selectivity for the vasculature is less than those of dihydropyridine derivatives, but this may be improved by a better understanding of the structure-activity relationship of tetrahydroisoquinolines and their interaction at L-type calcium channels. This could be accomplished, for instance, by further studies of the effects of substituted tetrahydroisoquinolines on t3H]nitrendipine binding (8). Acknowledgments HD is a AHFMR fellow. We thank Prof. S. X. Peng at the China Pharmaceutical University for kindly supplying CPU-23.

Cardiovascular Drug Reviews, Vol. 14, N o . 4 , 1996

CPU-23

379

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Cardiovascular Drug Reviews, Vol. 14. No. 4 , 1996